Single immunization with a recombinant multiple-epitope protein induced protection against FMDV type Asia 1 in cattle

To develop recombinant epitope vaccines against the foot-and-mouth disease virus (FMDV) serotype Asia 1, genes encoding six recombinant proteins (A1–A6) consisting of different combinations of B-cell and T-cell epitopes from VP1 capsid protein (VP1) of FMDV were constructed. These proteins were expressed in Escherichia coli and used to immunize animals. Our results showed that A6 elicited higher titers of neutralizing antibodies after single inoculation in guinea pigs than did the other five recombinant proteins, as determined by micro-neutralization tests. In addition, a strong lymphocyte proliferation response and Th1 type immunity were observed in splenocytes from the mice immunized with A6. Further tests carried out in cattle demonstrated that a single inoculation with A6 generated comparable levels of neutralizing antibodies as inactivated vaccine and protected 4 of 5 cattle against challenge with FMDV type Asia 1. Our results suggest that A6 might be a promising recombinant vaccine against FMDV type Asia 1 in cattle.     

The potential of retro-inverso peptides as synthetic vaccines

Retro-inverso peptides, also known as all-D-retro or retro-enantio peptides, are composed of D-amino acids assembled in the reverse order from that of the parent L-sequence. Since the orientation of the side-chains in a retro-inverso analogue is very similar to that in the parent L-peptide, this leads to a high level of antigenic cross-reactivity between the two peptides. The potential of retro-inverso peptides as synthetic vaccines has been investigated in the case of foot-and-mouth disease. A single inoculation of retro-inverso peptide corresponding to residues 141 - 159 of the VP1 protein of foot-and-mouth disease virus induced longer-lasting and higher antibody titres in immunised animals than the corresponding L-peptides. The antibodies cross-reacted strongly with virus particles and with L-peptides and conferred substantial protection in guinea-pigs challenged with the cognate virus. Retro-inverso peptides have considerable potential as synthetic vaccines, since their increased resistance to proteases may overcome one of the major drawbacks of classical L-peptide vaccines.     

静脉注射免疫球蛋白对呼吸道合胞病毒的清除作用

本文使用静脉注射免疫球蛋白（IVIG）0.4-0.5g/kg.d×2治疗23例呼吸道合胞病毒（RSV）感染病儿，提示IVIG能迅速提高血清IgG水平，补充大量RSV-sIgG中和抗体，有加速呼吸道中RSV清除的作用。     

Enhanced protective efficacy and reduced viral load of foot-and-mouth disease DNA vaccine with co-stimulatory molecules as the molecular adjuvants

To improve efficacy of DNA vaccination, various approaches have been developed, including the use of plasmid expressing co-stimulatory molecules as molecular adjuvants. In this study, we investigated whether co-inoculation of a construct expressing either 4-1BBL or OX40L as the molecular adjuvant with FMDV DNA vaccine, pcD-VP1, can increase immune responses and protective efficacies. Compared to the group immunized with pcD-VP1 alone, the co-inoculation of either molecular adjuvant induced a higher ratio of IgG2a/IgG1, higher levels of expression of IFN-gamma in CD4(+) and CD8(+) T cells and antigen-specific CTL responses, and more importantly provided an enhanced protection against the live FMDV challenge in animals. Concurrently, 4-1BBL as the molecular adjuvant dramatically reduced the viral loads of FMDV in vivo after the challenge. Together, the results demonstrate that co-stimulatory molecules 4-1BBL and OX40L can enhance the antigen-specific cell-mediated responses elicited by VP1 DNA vaccine and provide an enhanced protective efficacy with the reduced viral loads.     

基于流感病毒保守抗原的广谱流感疫苗研究进展

流感是由流感病毒感染引起的严重急性呼吸道传染病,接种流感疫苗是预防流感最经济、安全且有效的措施。目前广泛应用的传统流感疫苗的保护效果受到疫苗株与流行株表面抗原匹配程度的明显影响,难以有效应对因流感病毒发生抗原漂移或抗原转换而产生的无法预料的流行或大流行。因而开发能够诱导广谱和持久免疫的广谱流感疫苗是新型流感疫苗研发的重要方向。流感病毒基质、核蛋白和血凝素茎部结构域作为流感病毒的保守抗原,是当前广谱流感疫苗的主要靶抗原。此文就近年来基于流感病毒保守抗原的广谱流感疫苗的抗原选择、免疫保护制剂、临床研究进展做一综述。     

Synthesis and characterization of Respiratory Syncytial Virus protein G related peptides containing two disulfide bridges

Abstract: Respiratory Syncytial Virus (RSV) is the most important cause of bronchiolitis and viral pneumonia in infants and young children. Approximately 100 000 children are hospitalized in the USA each year as a result of RSV infections. During the research and development of subunit human Respiratory Syncytial Virus vaccines (hRSV), we have produced numerous synthetic peptides and recombinant proteins containing the four cysteines of the highly conserved central region of the G attachment protein. For several of these disulfide‐containing peptides, all possible oxidized isomers were synthesized using various oxidation conditions and resulting in different ratios of isomers. Each isolated isomer was fully characterized by RP‐HPLC, FZCE and ES‐MS after purification by preparative RP‐HPLC. The different cysteine pairings were unambiguously established after enzymatic digestion, LC‐MS analysis and peptide microsequencing. These synthesis and analytical methods were developed for the characterization on one hand, of recombinant fusion protein BBG2Na which is currently being investigated in advanced clinical phases as a very promising vaccine candidate, and on the other hand, for peptides which were synthesized to be evaluated as conjugate vaccines or as immunochemical tools, after covalent coupling to carrier proteins. Furthermore, these studies allowed us to determine which of the different possible isomers was the most stable and probably the preferred form in native conditions. Finally, the different oxidation and analysis conditions, should be useful for disulfide pairing studies of other peptides and proteins having the same ‘xCxxCxxxxxCxxxCx’ framework, such as G proteins of non‐human RSV strains, developed by other groups as veterinary vaccine candidates for example.     

The potential of retro-inverso peptides as synthetic vaccines

Retro-inverso peptides, also known as all-D-retro or retro-enantio peptides, are composed of D-amino acids assembled in the reverse order from that of the parent L-sequence. Since the orientation of the side-chains in a retro-inverso analogue is very similar to that in the parent L-peptide, this leads to a high level of antigenic cross-reactivity between the two peptides. The potential of retro-inverso peptides as synthetic vaccines has been investigated in the case of foot-and-mouth disease. A single inoculation of retro-inverso peptide corresponding to residues 141-159 of the VP1 protein of foot-and-mouth disease virus induced longer-lasting and higher antibody titres in immunised animals than the corresponding L-peptides. The antibodies cross-reacted strongly with virus particles and with L-peptides and conferred substantial protection in guinea-pigs challenged with the cognate virus. Retro-inverso peptides have considerable potential as synthetic vaccines, since their increased resistance to proteases may overcome one of the major drawbacks of classical L-peptide vaccines.     

流感病毒基质蛋白2胞外域作为广谱流感疫苗靶点研究现状

流感是由流感病毒引起的呼吸系统疾病,接种流感疫苗是预防流感的重要手段。疫苗的主要抗原是流感病毒表面蛋白血凝素和神经氨酸酶,但这两者都存在抗原漂移的现象,给疫苗设计带来巨大困难。广谱流感疫苗的候选抗原之一是高度保守的流感病毒基质蛋白2胞外域（matrix protein 2 ectodomain,M2e）。M2e在天然感染的情况下免疫原性较弱,但是可通过相关的佐剂、包裹材料和蛋白疫苗提高免疫原性。此文介绍了加强M2e免疫效果方式的研究以及M2e疫苗的临床研究进展。     

Evaluation of two carrier protein-angiotensin I conjugate vaccines to assess their future potential to control high blood pressure (hypertension) in man

AIMS: We aim to modulate the renin-angiotensin system (RAS) by active immunization against angiotensin I hormone (AI), potentially providing a novel conjugate vaccine treatment for hypertension in man. METHODS: Immunization studies in rat and human subjects compare the effectiveness of tetanus toxoid (TT) and keyhole limpet haemocyanin (KLH) vaccines for immunotherapy following conjugation with an AI peptide analogue (AI). Cardiovascular responses were assessed in immunized rats and human subjects (two-dose trial only), following increasing i.v. infusions of either AI or angiotensin II hormone (AII). RESULTS: The AI-TT and AI-KLH conjugate vaccines induced an equivalent immune response, and inhibition of the pressor effects to exogenous AI in rats. Single-dose clinical trials with both conjugate vaccines only resulted in an immune response to the KLH carrier protein. A two-dose clinical trial of AI-KLH conjugate vaccine resulted in a significant immune response to AI. A shift in diastolic blood pressure (DBP) dose-response was demonstrated following challenge with AI and AII for the study volunteer showing the largest anti-AI IgG induction. CONCLUSION: KLH was shown to be a suitable alternative to TT as a carrier protein for AI, thus supporting continued evaluation of our AI-KLH conjugate vaccine for treatment of hypertension in man.     

Rhinovirus infections and immunisation induce cross-serotype reactive antibodies to VP1

Rhinoviruses (RVs) are ubiquitous human respiratory viruses, the major cause of common colds, acute exacerbations of asthma and other respiratory diseases. The development of antibodies to RV following primary infection is poorly understood and there is currently no RV vaccine available. We therefore used mouse models of intranasal RV infection and immunisation to determine the induction, magnitude and specificity of antibody responses. Strong cross-serotype RV-specific IgG responses in serum and bronchoalveolar lavage were induced towards the RV capsid protein VP1. IgA responses were weaker, requiring two infections to generate detectable RV-specific binding. Similarly two or more RV infections were necessary to induce neutralising antibodies. Immunisation strategies boosted homotypic as well as inducing cross-serotype neutralising IgG responses. We conclude that VP1 based antigens combined with adjuvants may permit successful antibody-mediated vaccine design and development.     

The humoral response to Gardasil over four years as defined by total IgG and competitive Luminex immunoassay

Safe and effective vaccines against anogenital human papillomaviruses (HPV) are now available. These vaccines, composed of virus-like particles (VLPs) made from the L1 major capsid protein of specific HPV types, induce a polyclonal antibody response directed against specific conformational and linear epitopes displayed on the VLP. Numerous studies indicated the importance of neutralizing antibodies in protection from infection. However, our understanding of the antibody responses to these vaccines is not complete, and there is no established immune correlate of protection nor antibody threshold that correlates with protection against HPV infection or disease. In the current study, antibody responses of young women to Gardasil?, the quadrivalent HPV 6, 11, 16 and 18 L1 VLP vaccine (qHPV), were assessed through 48 months (M) in total IgG and competitive Luminex immunoassays (total IgG LIA and cLIA). The total IgG LIA was developed as a research assay to evaluate preclinical multivalent HPV VLP vaccine formulations. The cLIA simultaneously evaluates the antibody response to a unique conformational, neutralizing epitope on each of the four HPV types present in the quadrivalent vaccine; HPV 6, 11, 16 and 18. The same sera from women vaccinated with the qHPV vaccine were tested in both the total IgG LIA and the cLIA assays. The proportion of vaccinated women achieving seropositivity and the anti-HPV VLP total IgG and cLIA geometric mean titers (GMTs) were summarized at M7, M24, M48 based on the serostatus cut-points defined for each immunoassay. Overall, greater than 99% of subjects seroconverted to all four vaccine types in both assays; GMTs peaked at M7. For all four HPV types, regardless of the immunoassay used, the most significant decline in GMTs was observed between M7 and M24. By M24, the antibody titers had reached a plateau and minimal declines in antibody titers were observed between M24 and M48 for all four HPV types in both immunoassays. Testing the same sera, seropositivity for M48 HPV18 remained high (96.7%) in the total IgG LIA, but was 64.8% in the cLIA. The current study illustrates potential important differences in serologic assays utilized in the clinical trials of the two currently available HPV VLP vaccines (quadrivalent and bivalent). Differences in seropositivity status are attributed to the measurement parameters and sensitivity of the individual immunoassays and do not indicate reduced anti-HPV18 protective antibodies.     

A DNA vaccination regime including protein boost and electroporation protects cattle against foot-and-mouth disease

Protection against foot-and-mouth disease (FMD) using DNA technology has been documented for sheep and pigs but not for the highly susceptible species of cattle. Twenty-five Holstein Friesian cross-bred cattle were vaccinated twice, 21 days apart, with a DNA vaccine containing the capsid coding region (P1) along with the non-structural proteins 2A, 3C and 3D (pcDNA3.1/P1-2A3C3D) of O(1) Kaufbeuren alone or coated onto PLG (d,l-lactide-co-glycolide) microparticles. In some pcDNA3.1/P1-2A3C3D was also combined with an adjuvant plasmid expressing bovine granulocyte macrophage colony stimulating factor (GM-CSF). DNA vaccinations were administered intramuscularly with, or without, the use of electroporation and at 42 days post primary vaccination cattle received a protein boost of 146S FMD virus (FMDV) antigen and non-structural protein 3D. For comparison, four cattle were vaccinated with a conventional FMD vaccine and two more included as unvaccinated controls. Apart from those immunised with PLG microparticles all cattle were challenged with 10(5) TCID(50) cattle adapted O(1) Lausanne FMDV virus at day 93 post primary vaccination. All DNA vaccinated cattle regardless of regime developed good humoral and cell mediated responses prior to challenge. The best overall virus neutralising antibody, IFN-γ and clinical protection (75%) were seen in the cattle whereby the DNA was delivered by electroporation. In contrast, only 25% of cattle vaccinated with the DNA vaccine without electroporation were clinically protected. The addition of GM-CSF in combination with electroporation further improved the efficacy of the vaccine, as demonstrated from the reduction of clinical disease and virus excretions in nasal swabs. We thus demonstrate for the first time that cattle can be clinically protected against FMDV challenge following a DNA prime-protein boost strategy, and particularly when DNA vaccine is combined with GM-CSF and delivered by electroporation.     

Emerging human papillomavirus vaccines

Introduction: Identification of human papillomavirus (HPV) as the etiologic factor of cervical, anogenital, and a subset of head and neck cancers has stimulated the development of preventive and therapeutic HPV vaccines to control HPV-associated malignancies. Excitement has been generated by the commercialization of two preventive L1-based vaccines, which use HPV virus-like particles (VLPs) to generate capsid-specific neutralizing antibodies. However, factors such as high cost and requirement for cold chain have prevented widespread implementation where they are needed most.

Areas covered: Next generation preventive HPV vaccine candidates have focused on cost-effective stable alternatives and generating broader protection via targeting multivalent L1 VLPs, L2 capsid protein, and chimeric L1/L2 VLPs. Therapeutic HPV vaccine candidates have focused on enhancing T cell-mediated killing of HPV-transformed tumor cells, which constitutively express HPV-encoded proteins, E6 and E7. Several therapeutic HPV vaccines are in clinical trials.

Expert opinion: Although progress is being made, cost remains an issue inhibiting the use of preventive HPV vaccines in countries that carry the majority of the cervical cancer burden. In addition, progression of therapeutic HPV vaccines through clinical trials may require combination strategies employing different therapeutic modalities. As research in the development of HPV vaccines continues, we may generate effective strategies to control HPV-associated malignancies.     

DNA immunization with 2C FMDV non-structural protein reveals the presence of an immunodominant CD8+, CTL epitope for Balb/c mice

Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-γ) induction in CD8⁺, CD44^hi splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-γ production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses.     

Influenza virosome/DNA vaccine complex as a new formulation to induce intra-subtypic protection against influenza virus challenge

Influenza virosome is one of the commercially available vaccines that have been used for a number of years. Like other influenza vaccines, the efficacy of the virosomal vaccine is significantly compromised when circulating viruses do not have a good match with vaccine strains due to antigenic drift or less frequent emergence of a pandemic virus. A major advantage of virosome over other influenza vaccine platforms is its intrinsic adjuvant activity and potential carrier capability which have been exploited in this study to broaden vaccine protectivity by incorporating a conserved component of influenza virus in seasonal vaccine formulation. Influenza nucleoprotein (NP)-encoding plasmid was adsorbed onto surface of influenza virosomes as a virosome/DNA vaccine complex. Mice were immunized with a single dose of the influenza virosome attached with the NP plasmid or NP plasmid alone where both influenza virosomes and NP gene were derived from influenza A virus H1N1 New/Caledonia strain. Analysis of the cellular immune responses showed that 5μg (10-fold reduced dose) of the NP plasmid attached to the virosomes induced T cell responses equivalent to those elicited by 50μg of NP plasmid alone as assessed by IFN-γ and granzyme B ELISPOT. Furthermore, the influenza virosome/NP plasmid complex protected mice against intra-subtypic challenge with the mouse adapted H1N1 PR8 virus, while mice immunized with the virosome alone did not survive. Results of hemagglutination inhibition test showed that the observed intra-subtypic cross-protection could not be attributed to neutralizing antibodies. These findings suggest that influenza virosomes could be equipped with an NP-encoding plasmid in a dose-sparing fashion to elicit anti-influenza cytotoxic immune responses and broaden the vaccine coverage against antigenic drift.     

Investigation in a model system of the effects of combinations of anthrax and pertussis vaccines administered to service personnel in the 1991 Gulf War

The toxicity and immunogenicity of the anthrax and pertussis vaccine combinations used in the 1991 Gulf War was assessed in NIH, A/J and Balb/c mice. Inoculation of pertussis vaccines, vaccine combinations, or aluminium salt caused illness, splenomegaly and significant weight loss. Although some animals recovered eventually, a lethal form of ascites developed in some NIH mice and body weights of A/J and Balb/c mice remained below normal levels. Inoculation of anthrax vaccine produced little effect. Exposure to diluted vaccine combinations produced less serious side effects of shorter duration. Single vaccinations induced specific IgG1 antibodies whereas a mixture of IgG1 and IgG2a was produced after multiple injections. Antigen stimulation of spleen cells from mice exposed to pertussis vaccines induced high levels of NO and IL-6, whereas stimulated spleen cells from mice exposed to anthrax vaccine produced only low levels of IL-6. In mice, pertussis vaccines act as an adjuvant for anthrax vaccine, but these vaccines are also the major cause of toxicity of the vaccine combination. The relatively high vaccine dose used, together with the low sensitivity of mice to anthrax toxin, emphasises that caution should be exercised in applying these results to human recipients of these vaccines.     

Measurement over 1 Year of Neutralizing Antibodies in Cattle Immunized with Trivalent Vaccines Recombinant Alpha,Beta and Epsilon of Clostridium perfringens

The alpha (CPA), beta (CPB) and epsilon (ETX) toxins of Clostridium perfringens are responsible for causing diseases that are difficult to eradicate and have lethal potential in production animals. Vaccination of herds is still the best control strategy. Recombinant clostridial vaccines have shown good success at inducing neutralizing antibody titers and appear to be a viable alternative to the conventional production of commercial clostridial toxoids. Research is still needed on the longevity of the humoral immune response induced by recombinant proteins in immunized animals, preferably in target species. The objective of this study was to measure the humoral immune response of cattle immunized with trivalent vaccines containing the recombinant proteins alpha (rCPA), beta (rCPB) and epsilon (rETX) of C. perfringens produced in Escherichia coli at three different concentrations (100, 200, and 400 µg) of each protein for 12 months. The recombinant vaccines containing 200 (RV2) and 400 µg (RV3) yielded statistically similar results at 56 days. They performed better throughout the study period because they induced higher neutralizing antibody titers and were detectable for up to 150 and 180 days, respectively. Regarding industrial-scale production, RV2 would be the most economical and viable formulation as it achieved results similar to RV3 at half the concentration of recombinant proteins in its formulation. However, none of the vaccines tested induced the production of detectable antibody titers on day 365 of the experiment, the time of revaccination typically recommended in vaccination protocols. Thus, reiterating the need for research in the field of vaccinology to achieve greater longevity of the humoral immune response against these clostridial toxins in animals, in addition to the need to discuss the vaccine schedules and protocols adopted in cattle production.     

Expression of Recombinant Rotavirus Proteins Harboring Antigenic Epitopes of the Hepatitis A Virus Polyprotein in Insect Cells

Rotavirus and hepatitis A virus (HAV) spread by the fecal-oral route and infections are important in public health, especially in developing countries. Here, two antigenic epitopes of the HAV polyprotein, domain 2 (D2) and domain 3 (D3), were recombined with rotavirus VP7, generating D2/VP7 and D3/VP7, cloned in a baculovirus expression system, and expressed in Spodoptera frugiperda 9 (Sf9) insect cells. All were highly expressed, with peak expression 2 days post-infection. Western blotting and ELISA revealed that two chimeric proteins were antigenic, but only D2/VP7 was immunogenic and elicited neutralizing antibody responses against rotavirus and HAV by neutralization assay, implicating D2/VP7 as a multivalent subunit-vaccine Candidate for preventing both rotavirus and HAV infections.     

Single-shot AAV-vectored vaccine against SARS-CoV-2 with fast and long-lasting immunity

Due to the insufficient long-term protection and significant efficacy reduction to new variants of current COVID-19 vaccines, the epidemic prevention and control are still challenging. Here, we employ a capsid and antigen structure engineering (CASE) strategy to manufacture an adeno-associated viral serotype 6-based vaccine (S663V-RBD), which expresses trimeric receptor binding domain (RBD) of spike protein fused with a biological adjuvant RS09. Impressively, the engineered S663V-RBD could rapidly induce a satisfactory RBD-specific IgG titer within 2 weeks and maintain the titer for more than 4 months. Compared to the licensed BBIBP-CorV (Sinopharm, China), a single-dose S663V-RBD induced more endurable and robust immune responses in mice and elicited superior neutralizing antibodies against three typical SARS-CoV-2 pseudoviruses including wild type, C.37 (Lambda) and B.1.617.2 (Delta). More interestingly, the intramuscular injection of S663V-RBD could overcome pre-existing immunity against the capsid. Given its effectiveness, the CASE-based S663V-RBD may provide a new solution for the current and next pandemic.     

DNA immunization with 2C FMDV non-structural protein reveals the presence of an immunodominant CD8+, CTL epitope for Balb/c mice

Outbreaks of foot and mouth disease virus (FMDV) have devastating economic consequences in affected areas. The presence of multiple serotypes and virus variants makes vaccination complicated. A better understanding of protective immune mechanisms may help in development of novel vaccines with cross protective capacity. While much attention has been devoted to humoral responses to FMDV, less is known about the role of cell-mediated responses in protective immunity. Predictions of potential CTL epitopes by two different computer algorithms identified the viral 2C protein as containing a potential murine H2-Kd CTL epitope located in its amino-terminal half. DNA vaccination of mice with a plasmid expressing the 2C protein and a fragment thereof confirmed that this was indeed a CTL epitope, as shown by interferon gamma (IFN-gamma) induction in CD8+, CD44(hi) splenocytes after in vitro stimulation with peptides containing the amino acid sequence KYKDAKEWL, predicted for the CTL epitope. A peptide with the variant sequence KYKEAKEWL induced similar responses, indicating tolerability towards a conservative substitution at the altered residue. Virus infection likewise induced a measurable CTL response against KYKDAKEWL, although less clear due to a higher background of IFN-gamma production in splenocytes from infected mice. Challenge of vaccinated mice showed that the CTL response induced by the 2C protein was not protective, since viremia and mortality were unaffected by vaccination. The implications for vaccine development are discussed in the context of cross-serotype reactive responses.